These are the answers I received on my posting regarding southwestern
blotting. I hope they are useful, thanks to the people who responded.
Beatriz.
From: marka at ATHENA.MIT.EDU
I did some SW blotting a couple of years ago as part of a lab class at Cambridge.
Jean Thomas gave the class, and I think she developed some of the methods, if
not I am certain she has used them in her work on histones. A literature search
on her would probably give you some relevant papers.
I'll try & ressurect my old lab notes. If I don't get back to you & you are still
in need of help let me know.
MArk.
From: MANGALAM at SALK-SC2.SDSC.EDU
Hi,
As one who has used and abused Southwesterns for years, I can send you the
protocol that seems to have
worked for me. [stuff deleted] I've used both denaturing and
non-denaturing gels, but prefer the denaturing because of the resolution of the
bands and the sizing. Certainly, if the proein of interest is irreversibly
bunged by denaturing, then you're out of luck but it is amazing what a protein
can go thru and still bind DNA in a relatively specific manner. So far, I've
used the SW to detect homeobox, POU-homeobox, HMG box proteins, as well as
countless other (unidentified) proteins in nuclear extracts. You can also
use mutant probes to discern between specific and non-specific binding (or
increase the amount of non-specific competitor DNA.
More recently, I and others have been trying to use variants of this to
trap or identify co-factors that associate with DNA binders. This variation
(obviously not perfected yet) uses the standard SW technique, except that the
binding site is not labeled and labeled cofactor is added to the hybe mix.
Or vice versa.
In fact the SW is not very new, but is (I think) very underutilized.
The main problem is that it is a lot more trouble than a gel shift, but it
gives you more specific information.
The first references I found are:
Bowen et al 1980
NAR 8:1-8
and
Jack et al 1982
Cold Spring Harbor Symp. Quant Biol 68:483-491
Let me know if you need anything else
Harry Mangalam
MNL-T
Salk Institute
619 453-4100, x360
Oops: to answer your remaining questions... Larger fragments are better as
probes, especiallt if you can ligate individual binding sites together to
form multimers. I block with milk unless the blot is to be probed with RNA
(yes, a Northwestern) in which case I block with BSA and/or ovalbumin. I use
nitrocellulose for the support but I see no reason why the newer nylon-based
supports would not work.
Cheers
From: TJAMES at EAGLE.WESLEYAN.EDU
X-VMS-News: eagle.wesleyan.edu bionet.molbio.methds-reagnts:480
> I would like to know if anybody can supply me with a reference or
> protocol for Southwestern blotting. It is supposed to be a new (to
> me) technique in which you separate your proteins by gel
> electrophoresis and probe with DNA. This would be very similar to a
etc.
Southwestern blotting has been around for some time. There is paper by
Weintraub and some one back in 1984 may be. I cannot find the exact reference.
But I can find it if you haven't already got. Many of the earlier experiments
were done with SDS gels but lately more non-denaturing gels have also been
used. It has some limitations. Most modern experimenters prefer to use gel
mobility shift assays instead. You can use Nitrocellulose or Nylon for SW. You
should use an effective blocking agent. I have not used milk powder but 2% BSA
works fine. Incubations should be in 50-100 mM salt concentrations initially
but then you can try increasing it. You should certainly run controls with
proteins that you know will not bind to your DNA.
--
Tharappel C. James
Department of Molecular Biology
Wesleyan University
From: Robert Kucharski <KUCHARZ at PLEARN>
Dear Beatriz,
I can send you some Southwestern protocols,but in two or three weeks,if you
are still looking for them.There were a review article on basic Southwestern
techniques in Biotechniques,I think,but I don't remember all references.
Now I can only say,that:
1.You can try Laemmli denaturing gels
2.Small probes with many recognition sites (concatamers) are preferable
3.You can use nonfat milk to block filters
4.You can use nitrocellulose but the transfer must be performed in appropriate
buffer (like Laemmli buffer:192mM glycine,25mM Tris(base)) with 20% methanol.
If you want to receive detailed references and\or protocols,write directly to
me (but I would be able to answer after August,25).
Robert Kucharski.